Separation and Characterization of the Activated Pool of Colony-Stimulating Factor 1 Receptor Forming Distinct Multimeric Complexes with Signalling Molecules in Macrophages

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.     

Structural features of the colony-stimulating factor 1 receptor that affect its association with phosphatidylinositol 3-kinase.

The colony-stimulating factor 1 receptor (CSF-1R), immunoprecipitated with either anti-phosphotyrosine or anti-receptor antibodies from lysates of ligand-stimulated cells, is associated with a phosphatidylinositol (PtdIns) 3-kinase activity. The ligand-independent transforming efficiencies of human CSF-1R mutants containing certain amino acid substitutions at codon 301 in their extracellular domains correlated directly with their levels of associated lipid kinase activity. A tyrosine kinase defective CSF-1R mutant (CSF-1R[met616]), containing a mutated ATP binding site, lacked associated PtdIns 3-kinase activity in immune complexes recovered from CSF-1-stimulated cells. However, CSF-1R[met616] associated with PtdIns 3-kinase when phosphorylated in trans in CSF-1-stimulated cells coexpressing an enzymatically competent CSF-1R tyrosine kinase. Another CSF-1R mutant, (CSF-1R[delta KI]), lacking 67 amino acids from its intracellular 'kinase insert' domain, exhibited a partially impaired ligand-dependent mitogenic response and a significant reduction in its associated PtdIns 3-kinase activity. Ligand-stimulated CSF-1R[delta KI] molecules contained levels of phosphotyrosine almost equivalent to wild-type receptors, but were phosphorylated at different sites in vitro. Therefore, the association of CSF-1R with active PtdIns 3-kinase required the receptor tyrosine kinase activity, was triggered by receptor phosphorylation on tyrosine and, in this series of mutants, correlated with their mitogenic potential. Although the receptor KI domain strongly contributes to the association with PtdIns 3-kinase, this region is not strictly essential for the interaction.     

Structural basis for the dual recognition of helical cytokines IL-34 and CSF-1 by CSF-1R

Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.     

Peptide antisera to human colony-stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase.

A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and -KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti-C-ter serum inhibited binding. We conclude that (i) only a minority of ligand-activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site.     

The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1

Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.     

A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential

A human CSF-1 receptor containing an "activating" mutation in its extracellular domain (serine for leucine 301) induced morphologic transformation, anchorage-independent growth, and tumorigenicity in mouse NIH 3T3 cells. A second regulatory mutation within the receptor's intracytoplasmic carboxy-terminal tail (phenylalanine for tyrosine 969) augmented transforming efficiency but was itself insufficient to induce transformation. Like the v-fms oncogene product, receptors bearing the activating mutation retained high-affinity binding sites for CSF-1 but were retarded in transport to the cell surface and were phosphorylated on tyrosine in the absence of ligand. Although the activating mutation does not affect the CSF-1 binding site in the receptor extracellular domain, it must induce a conformational change that mimics the effect of ligand binding, resulting in CSF-1-independent signals for cell growth.     

A proteomics strategy for the enrichment of receptor-associated complexes

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.     

Receptor-type Protein-tyrosine Phosphatase ζ Is a Functional Receptor for Interleukin-34

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.     

The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation.

Colony-stimulating factor-1 (CSF-1) activation of the CSF-1 receptor (CSF-1R) causes Cbl protooncoprotein tyrosine phosphorylation, Cbl-CSF-1R association and their simultaneous multiubiquitination at the plasma membrane. The CSF-1R is then rapidly internalized and degraded, whereas Cbl is deubiquitinated in the cytoplasm without being degraded. We have used primary macrophages from gene-targeted mice to study the role of Cbl. Cbl-/- macrophages form denser colonies and, at limiting CSF-1 concentrations, proliferate faster than Cbl+/+ macrophages. Their CSF-1Rs fail to exhibit multiubiquitination and a second wave of tyrosine phosphorylation previously suggested to be involved in preparation of the CSF-1-CSF-1R complex for endocytosis. Consistent with this result, Cbl-/- macrophage cell surface CSF-1-CSF-1R complexes are internalized more slowly, yet are still lysosomally degraded, and the CSF-1 utilization by Cbl-/- macrophages is reduced approximately 2-fold. Thus, attenuation of proliferation by Cbl is associated with its positive regulation of the coordinated multiubiquitination and endocytosis of the activated CSF-1R, and a reduction in the time that the CSF-1R signals from the cell surface. The results provide a paradigm for studies of the mechanisms underlying Cbl attenuation of proliferative responses induced by ligation of receptor tyrosine kinases.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.     

Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms.

The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.     

Antibody-induced mitogenicity mediated by a chimeric CD2-c-fms receptor.

A chimeric receptor composed of the extracellular domain of the human T-cell antigen CD2 (T11) joined to the membrane-spanning segment and the intracellular tyrosine kinase domain of the human colony-stimulating factor 1 receptor (CSF-1R) was expressed in murine NIH 3T3 fibroblasts. Stimulation of these cells with monoclonal antibodies to CD2 induced phosphorylation of the chimeric glycoprotein on tyrosine, receptor downmodulation, and mitogenesis. In contrast, neither human CSF-1R nor the chimeric receptor was able to function in interleukin-2-dependent murine T cells. In fibroblasts, then, CSF-1 per se is not required for activation of the receptor kinase or for a biological response, whereas in T cells, CSF-1R may be unable to engage the downstream signal transduction machinery.     

Cloning and expression of feline colony stimulating factor receptor (CSF-1R) and analysis of the species specificity of stimulation by colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34)

Colony stimulating factor (CSF-1) and its receptor, CSF-1R, have been previously well studied in humans and rodents to dissect the role they play in development of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, IL-34 has been described in several species. In this study, we have cloned and expressed the feline CSF-1R and examined the responsiveness to CSF-1 and IL-34 from a range of species. The results indicate that pig and human CSF-1 and human IL-34 are equally effective in cats, where both mouse CSF-1 and IL-34 are significantly less active. Recombinant human CSF-1 can be used to generate populations of feline bone marrow and monocyte derived macrophages that can be used to further dissect macrophage-specific gene expression in this species, and to compare it to data derived from mouse, human and pig. These results set the scene for therapeutic use of CSF-1 and IL-34 in cats.     

Mapping mouse IL-13 binding regions using structure modeling, molecular docking, and high-density peptide microarray analysis

Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also used by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.     

Mouse mesangial cells produce colony-stimulating factor-1 (CSF-1) and express the CSF-1 receptor

CSF-1 stimulates the survival, proliferation, and differentiation of mononuclear phagocytes and may also play a role in placental development. The expression of CSF-1 and the CSF-1 receptor (CSF-1R) and their regulation were examined in cultures of mouse mesangial cells (MC). The concentration of CSF-1 in the medium of cultured MC increased linearly with time over 24 h. IFN-gamma stimulated and dibutyryl cyclic AMP inhibited CSF-1 production in a dose-dependent manner. MC expression of CSF-1 mRNA was shown by Northern blot analysis, and CSF-1 mRNA levels were increased within 4 h of IFN-gamma addition and inhibited within 4 h of dibutyryl cyclic AMP addition. Indirect immunofluorescence indicated that 90% of the untreated cultured MC expressed CSF-1. In addition, CSF-1R expression by MC was demonstrated by immunofluorescence with anti-receptor antibody, specific binding of [125I] CSF-1, and expression of the CSF-1R mRNA by Northern blot analysis. Thus, mouse MC, specialized pericytes of non-bone marrow origin, not only produce CSF-1 but also express receptors for CSF-1. The effects of CSF-1 on MC may be important in the control of immune function in the glomerulus.